Hepatic guanylate cyclase was further purified and characterized. The most highly purified preparation had a specific activity of 0.9 to 1.3 micron mol/mg-min, S20w of 6.38, Stokes radius of 47.1 A, and molecular weight of 133,000 daltons with a monomer molecular weight of 69,000 daltons. The products of the guanylate cyclase reaction, cGMP and PPi were definitely identified and quantitated by 31 P NMR. An activator of guanylate cyclase from rat liver was further purified and characterized.